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Chapter 5
General Aspects of Ozone Therapy
Ruhi CakirAdditional information is available at the end of the
chapter
http://dx.doi.org/10.5772/57470
1. IntroductionOzone is a natural gas consisting of three oxygen
atoms that has a distinctive odor. It isintrinsically hazardous
over tolerable doses for living organisms. Ozone therapy is a
medicaltherapy that a mixture of oxygen and ozone which is called
medical ozone is used as a medicaldrug, more correctly pro-drug.
Medical ozone contains less than 5% of ozone at
maximumconcentration where rest of it is pure medical oxygen.The
unbelievable versatility of ozone therapy is due to the cascade of
ozone-derived compounds able to act on several targets leading to a
multifactorial correction of various pathological states. Ozone
therapy can improve well-being and delay the negative effects of
aging.Aging process basicly related with oxidants and anti-oxidants
balance, Advanced GlycoslationEnd Substances (AGEs), role of genes
and immune system, relevance of telomeres andtelomerase, hormones,
nutrition, environmental factors and some other factors.
2. What is ozone therapy?Ozone therapy is a general termination
of a medical therapy that medical ozone gas is used asdrug by
several methods. Some of these methods are systemic where many
others are localapplications. Ozonated autohaemotherapy (O3-AHT)
widely known by people firstly described by Wehrli and Steinbart
and since 1954 it has been used in millions of patients in
differentpathologies with apparent clinical benefit. AHT might be
applied in two forms, Major AHTsimply driving 100-150 ml of venous
blood into a sterile bottle made of neutral glass or otherozone
resistant material where blood and medical ozone is mixed in
therapeutic doses andthen reinfused back to the donor without side
effects. 3.13 % Natrium Citrate solution is usedas an anticoagulant
during the procedure with short lasting effect. In some patients
Heparinmight be used instead of Natrium Citrate depending on the
patients case.
2014 The Author(s). Licensee InTech. This chapter is distributed
under the terms of the Creative CommonsAttribution License
(http://creativecommons.org/licenses/by/3.0), which permits
unrestricted use,distribution, and reproduction in any medium,
provided the original work is properly cited.
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Figure 1. Schematic drawing of the components necessary to
perform major autohematherapy
Minor AHT is very similar to major AHT method with a few
differences, where 5-10 ml ofblood is mixed with precise dose of
medical ozone in a syringe and reinjected by intramuscularroute to
the donor that no anticoagulant is used. Rectal insufflation (RI)
of medical ozone gasis another method of systemic ozone therapy
that is applied on some cases if others methodscannot be done or
this method is preferred over others due to diseases.Ozone is
normally present as gas made of three atoms of oxygen with a cyclic
structure. Themedical generator of ozone produces it from pure
oxygen passing through a high voltagegradient (5-13 mV) according
to the reaction.
+ 2 3 3O 68,400cal 2O (1)
Ozone is 1.6 fold denser and 10-fold more soluble in water (49.0
mL in 100 mL water at 0_C)than oxygen. Although ozone is not a
radical molecule, it is the third most potent oxidant (E_512.076 V)
after fluorine and persulfate. Ozone is an unstable gas that cannot
be stored andshould be used at once because it has a half life of
40 min at 20_C
2.1. What is the behavior and fate of ozone after coming in
contact with body fluids?The essential concepts to bear in mind are
the following;a. As any other gas, ozone dissolves physically in
pure water according to Henrys Law in
relation to temperature, pressure and ozone concentration. Only
in this situation ozonedoes not react and in a tightly closed glass
bottle, the ozonated water is useful as adisinfectant that remains
active for a couple of days
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b. On the other hand, at variance with oxygen, ozone reacts
immediately as soon as it isdissolved in biological water
(physiological saline, plasma, lymph, urine)
+ + 3 2O biomolecules O O, (2)
Where atomic oxygen behaves as a very reactive atom. Contrary to
the incorrect belief thatozone penetrates through the skin and
mucosae or enters into the cells, it is emphasized that,after the
mentioned reaction, ozone does not exist any longer. In order of
preference, ozonereacts with polyunsaturated fatty acids (PUFA),
antioxidants such as ascorbic and uric acids,thiol compounds
with-SH groups such as cysteine, reduced glutathione (GSH) and
albumin.Depending upon the ozone dose, carbohydrates, enzymes, DNA
and RNA can also be affected.All of these compounds act as electron
donor and undergo oxidation.c. The main reaction:
- = - + + - = + - = + 3 2 2 2R CH CH R O H O R CH O R CH O H O
(3)
shows the simultaneous formation of one mole of hydrogen
peroxide (included among reactiveoxygen species, ROS) and of two
moles of lipid oxidation products (LOPs) [12].The fundamental ROS
molecule is hydrogen peroxide, which is a non-radical oxidant able
toact as an ozone Messenger responsible for eliciting several
biological and therapeutic effects[13,14]. The concept that ROS are
always harmful has been widely revised because, inphysiological
amounts, they act as regulators of signal transduction and
represent importantmediators of host defense and immune responses.
Presence of traces of Fe++should be avoidedbecause, in the presence
of hydrogen peroxide, via the Fentons reaction, they will catalyze
theformation of the most reactive OH, (hydroxyl radical).It is
determined [15] that the formation of nitrogen monoxide (NO,) in
human endothelial cellsexposed to ozonated serum. Attention should
be paid to the fact that an excess of ROS can leadto the formation
of other toxic compounds such as peroxynitrite (O=NOO-) and
hypochloriteanion (ClO-).Although ROS have a lifetime of less than
a second, they can damage crucial cell componentsand, therefore,
their generation must be precisely calibrated to achieve a
biological effectwithout any damage. This can be achieved by
regulating the ozone dose (ozone concentrationas mg/mL of gas per
mL of blood in 1:1 ratio) against the antioxidant capacity of blood
thatcan be measured and, if necessary, strengthened by oral
administration of antioxidants beforeand throughout ozone
therapy.d. LOPs production follows peroxidation of PUFA present in
the plasma: they are hetero
geneous and can be classified as lipoperoxides (LOO,), alkoxyl
radicals (LO,), lipohydroperoxides (LOOH), isoprostanes and
alkenals, among which are 4-hydroxy-2,3transnonenal (HNE) and
malonyldialdehyde (MDA). Radicals and aldehydes areintrinsically
toxic and must be generated in very low concentrations. They are in
vitro far
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more stable (6) than ROS but fortunately, upon blood reinfusion,
they undergo a markeddilution in body fluids, excretion (via urine
and bile), and metabolism by GSH-transferase(GSH-Tr) and aldehyde
dehydrogenases. Thus, only submicromolar concentrations canreach
all organs, particularly bone marrow, liver, central nervous system
(CNS), endocrineglands, etc., where they act as signaling molecules
of an ongoing acute oxidative stress[16].
If the stage of the disease is not too far advanced, these
molecules can elicit the upregulationof antioxidant enzymes such as
superoxide dismutase (SOD), GSH-peroxidases (GSH-Px),GSH-reductase
(GSH-Rd) and catalase (CAT). Interestingly, Iles and Liu [17] have
justdemonstrated that HNE, by inducing the expression of glutamate
cysteine ligase, causes anintracellular increase of GSH, which
plays a key role in antioxidant defense. Furthermore,LOPs induce
oxidative stress proteins, one of which is heme-oxygenase I (HO-1
or HSP-32)which, after breaking down the heme molecule, delivers
very useful compounds such as COand bilirubin [18]. Bilirubin is a
significant lipophilic antioxidant and a trace of CO cooperateswith
NO in regulating vasodilation by activating cyclic GMP. Fe++is
promptly chelated byupregulated ferritin. The induction of HO-1
after an oxidative stress has been described inhundreds of papers
as one of the most important antioxidant defense and protective
enzyme.Moreover, LOPs exert a neuroimmunomodulatory effect
highlighted by a feeling of well beingreported by patients during
ozone therapy.Although it remains hypothetical, it is possible that
LOP, throughout the treatments, acting asacute oxidative stressors
in the bone marrow microenvironments activate the release
ofmetalloproteinases, of which MP-9 particularly may favor the
detachment of staminal cells[11]. These cells, once in the blood
circulation, may be attracted and home at sites where aprevious
injury (a trauma or an ischemic-degenerative event) has taken
place. The potentialrelevance of such an event would have a huge
practical importance and will avoid theunnatural, costly and
scarcely effective practice of the bone marrow collection with the
needof the successive and uncertain reinfusion [19].It is
emphasized that submicromolar LOPs levels can be stimulatory and
beneficial, whereashigh levels can be toxic. This conclusion, based
on many experimental data [16], reinforces theconcept that optimal
ozone concentrations are critical for achieving a therapeutic
result: toolow concentrations are practically useless (at best
elicit a placebo effect), too high may elicit anegative effect
(malaise, fatigue) so that they must be just above the threshold
level to yield anacute, absolutely transitory oxidative stress
capable of triggering biological effects withouttoxicity. In
conclusion, it must be clear that the ozonation process either
happening in blood,or intradiscal or in an intramuscular site
represents an acute oxidative stress. However,provided that it is
precisely calculated according to a judicious ozone dosage, it is
not deleterious but is actually capable of eliciting a multitude of
useful biological responses and, possibly,can reverse a chronic
oxidative stress due to aging, chronic infections, diabetes,
atherosclerosis,degenerative processes and cancer. Indeed, the
ozonotherapeutic act is interpreted as an atoxicbut real
therapeutic shock able to restore homeostasis.
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2.2. Which are the biological effects elicited by ROS and
LOPs?The ozonation process is therefore characterized by the
formation of ROS and LOPs acting intwo phases. This process happens
either ex vivo (as a typical example in the blood collectedin a
glass bottle) or in vivo (after an intramuscular injection of
ozone) but while ROS are actingimmediately and disappear (early and
short-acting messengers), LOPs, via the circulation,distribute
throughout the tissues and eventually only a few molecules bind to
cell receptors.Their pharmacodynamics allow minimizing their
potential toxicity and allows them to becomelate and long-lasting
messengers.Formation of ROS in the plasma is extremely rapid and is
accompanied by a transitory andsmall ozone dosedependent decrease
(ranging from 5 to 25%) of the antioxidant capacity.Importantly,
this return to normal within 1520 min owes to the efficient
recycling of oxidizedcompounds such as dehydroascorbate to ascorbic
acid [20]. H2O2 diffuses easily from theplasma into the cells and
its sudden appearance in the cytoplasm represents the
triggeringstimulus: depending upon the cell type, different
biochemical pathways can be concurrentlyactivated in erythrocytes,
leukocytes and platelets resulting in numerous biological effects.
Itmust be noted that between the plasma and the cytoplasm
compartments there is a gradientand the intracellular H2O2
concentration is only about 1/10 of the plasmatic one [21]. The
rapidreduction to water is operated by the high concentration of
GSH, CAT and GSH-Px; nonetheless, H2O2 must be above the threshold
concentration for activating several biochemicalpathways.Let us now
examine how hydrogen peroxide, now universally recognized as one of
the mainintracellular signalling molecules [13], acts on the
different blood cells. The mass of erythrocytes mops up the bulk of
hydrogen peroxide: GSH is promptly oxidized to GSSG and the
cell,extremely sensitive to the reduction of the GSH/GSSG ratio,
immediately corrects the unbalance by either extruding GSSG or
reducing it with GSH-Rd at the expense of ascorbate or ofthe
reduced nicotinamide adenine dinucleotide phosphate (NADPH), which
serves as a crucialelectron donor. Next, the oxidized NADP is
reduced after the activation of the pentosephosphate pathway, of
which glucose-6-phosphate dehydrogenase (G-6PD) is the key
enzyme.It is determined that a small but significant increase of
ATP formation [10,11], but whether thisis due to the activation of
the pentose cycle or to phosphofructokinase or to both remains tobe
clarified. Moreover, for a brief period the reinfused erythrocytes
enhance the delivery ofoxygen into ischemic tissues because of a
shift to the right of the oxygenhemoglobin dissociation curve, due
either to a slight decrease of intracellular pH (Bohr effect)
or/and an increaseof 2,3-diphosphoglycerate (2,3-DPG) levels.
Obviously, one AHT treatment has a minimaleffect and we need to
ozonate at least 2.54 L of blood within a period of 3060 days.
Duringthis period, LOPs act as repeated stressors on the bone
marrow and these frequent stimulicause the adaptation to the ozone
stress during erythrogenesis with upregulation of antioxidant
enzymes. As a consequence, a patient with chronic limb ischemia
undergoing ozonetherapy can have a clinical improvement due to the
formation of successive cohorts oferythrocytes progressively more
capable of delivering oxygen to his/her ischemic tissues.However,
the final improvement is also due to the localized release of NO,
CO and growthfactors released from platelets and endothelial
cells.
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Although ozone is one of the most potent disinfectants, it
cannot inactivate bacteria, virusesand fungi in vivo because,
paradoxically, the pathogens are well protected, particularly
insidethe cells, by the powerful antioxidant system. Thus, as it
was proposed a long time ago [22,23],ozone acts as a mild enhancer
of the immune system by activating neutrophils and stimulatingthe
synthesis of some cytokines (2,57). Once again, the crucial
messenger is hydrogenperoxide, which after entering into the
cytoplasm of blood mononuclear cells (BMC) byoxidizing selected
cysteines, activates a tyrosine kinase, which then phosphorylates
thetranscription factor nuclear factor kB [24], allowing the
release of a heterodimer (p50+p65).This complex moves on to the
nucleus and switches on some hundred genes eventuallyresponsible
for causing the synthesis of several proteins, among which are the
acute-phasereactants and numerous interleukins. In the past, it was
measured the release of severalcytokines from ozonated blood upon
in vitro incubation (27). Once the ozonated leukocytesreturn to the
circulation, they home in lymphoid microenvironments and
successively releasecytokines acting in a paracrine fashion on
neighboring cells with a possible reactivation of adepressed immune
system [25]. This process, described as the physiological cytokine
response,is part of the innate immune system and helps us to
survive in a hostile environment.During ozonation of blood,
particularly if it is anticoagulated with heparin, we have noted
anozone dose-dependent increase of activation of platelets [8, 26]
with a consequent release oftypical growth factors, which will
enhance the healing of chronic ulcers in ischemic patients.Whenever
possible, the use of heparin as an anticoagulant is preferable to
sodium citratebecause, by not chelating plasmatic Ca++, it
reinforces biochemical and electric events.During reinfusion of the
ozonated blood into the donor, the vast expanse of the
endothelialcells will be activated by LOPs, resulting in an
increased production of NO, plasma S-nitrosothiols and
S-nitrosohemoglobin [15, 27]. Whereas NO has a half-life of less
than 1 sec,protein-bound-NO can exert vasodilation also at distant
ischemic vascular sites with relevanttherapeutic effect.Moreover,
on the basis of the phenomenon of ozone tolerance that says the
exposure of anorganism to a low level of an agent, harmful at high
levels, induces an adaptive and beneficialresponse [28,29], it is
postulated that LOPs, by acting as long-distance messengers, can
transmitto all organs the information of an acute oxidative stres
[10, 11]. The bone marrow is particularly relevant because it can
upregulate antioxidant enzymes during erythrogenesis and allowsthe
release of staminal cells for possibly regenerating infarcted
organs. Moreover, the stimulation of the endocrine and central
nervous systems may help to understand why most patientsduring
prolonged ozone therapy report a feeling of euphoria and wellness,
probably due toan improved metabolism as well as to an enhanced
hormonal or neurotransmitter release.The paradoxical concept that
ozone eventually induces an antioxidant response capable
ofreversing a chronic oxidative stress is common in the animal and
vegetal kingdom and thereis good experimental evidence [3034] that
this phenomenon is present in the animal andvegetal kingdom.
Moreover, it is already supported by findings of an increased level
ofantioxidant enzymes and HO-1 during ozone therapy [10,11]. It
also suggests that a judicioususe of ozone, in spite of acting as
an oxidant, enhances the antioxidant capacity, which
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represents the critical factor for overcoming chronic viral
infections, ischemia and celldegeneration.
3. A concise summary of biological effects observed after ozone
therapy3.1. ErythrocytesThese cells respond with an activation of
glycolysis due to activation of the pentose hosphatepathway. It is
found that increased adenosine triphosphate levels (from 13899/260
to 19689/232mM) in patients with age-related macular degeneration
(ARMD) (atrophic form) after atherapeutic cycle (14 sessions) of
O3-AHT.26 Moreover, Viebahn [27] reported the same effectin
athletes and elderly patients after rectal insufflation of O2_/O3.
Ozonation implies a smallbut consistent oxidation of GSH to
glutathione disulfide, and GSH reductase utilizes thereduced form
of the coenzyme nicotinamide adenine dinucleotide phosphate
supplied byG6PDH to reduce glutathione disulfide to GSH, which
indeed returns rapidly to the originallevel. [28] The increase of
2,3-diphosphoglycerate varies depending on the basic level in
ARMDpatients and only those who had a low level showed a marked
increase with therapy. Viebahn,[27] after a longer cycle of therapy
in elderly people, observed a significant increase. Anincrease of
2,3-diphosphoglycerate level in oxyhemoglobin shifts to the right
(p50 valueincreases); its dissociation curve implies an increased
delivery of O2 into the hypoxic tissues.The life-span of ozonated
99Tc-labeled erythrocytes and their uptake by liver and spleen
arecomparable with oxygenated erythrocytes.(3)A problem still under
study regards the generation of biochemically improved
erythrocytesduring prolonged ozone therapy. While ROS have an
extremely short life, LOPs, during thereinfusion of ozonated blood,
return into the donors circulation. While they are fairly stablein
vitro, they rapidly disappear from blood in vivo owing to
considerable dilution into bodyfluids, degradation by aldehyde
dehydrogenases, excretion into bile and urine, and uptake invarious
organs including bone marrow cells. This process is crucial for
explaining the mechanism of ozone tolerance: during erythrogenesis,
submicromolar LOP concentrations canupregulate the synthesis of
antioxidant enzymes and indeed, after appropriate densitygradient
separation, it is found that young (lighter) erythrocytes contain
more G6PDH thanolder (heavier) cells generated before the therapy.
[25] This result suggests that ozone therapyenhances the generation
of erythrocytes with improved metabolic characteristics, a sort
ofsupergifted erythrocytes able to correct hypoxia in vascular
diseases.
3.2. LeukocytesThese were the cells that were examined first as
it is hypothesized that ozone could act as anIFN-g inducer.[29]
Since then it is shown [30,31] that ozone behaves as a weak
(compared withmitogens) cytokine (such as tumor necrosis factor-a,
interleukin-2, interleukin-6, interleukin-8,transforming growth
factor-b [TGF-b]) inducer. Several studies [32 - 35] have confirmed
thatozone can stimulate bronchoalveolar cells to release
proinflammatory cytokines and eicosanoids. Thanks to parallel
progress in understanding the role of antioxidants and redox
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regulation of gene transcription, it has been clarified that,
among several signals, H2O2 is oneof the most significant cytokine
inducers.[36] As already mentioned, after ozonation H2O2freely
diffuses into the leukocyte cytoplasm and activates specific
protein kinases that, byphosphorylating IkB bound to the nuclear
factor-kB allows the migration of the transcriptionheterodimer
p50_/p65 into the nucleus where it activates gene expression.[37]
Obviously H2O2must reach a concentration able to activate the
kinase without being instantaneously reducedby intracellular
antioxidants. [38] Therefore the relevance of the response depends
on the levelsof H2O2, which can act as either life or death
signals. The fact that ozone can either be a toxicor a useful
signal depends on the minimal antioxidant capacity of the
respiratory tract liningfluid, whereas blood has a very potent
capacity. The data in fact indicate that too little ozone(hence
H2O2) is ineffective and too much (or too little antioxidants) can
be toxic. During recentyears it is addressed the following
questions: first, as ozone acts as a mild cytokine inducer,does
reinfusion of ozonated blood modify the plasma cytokine level in
vivo? Second, does theinduction of oxidative stress proteins,
particularly of heme-oxygenase I (HO-I), and ofadaptation to the
therapeutic oxidative stress have an immunomodulatory effect? And
third,can we devise an optimal schedule for improving the immune
reactivity in immunodepressedpatients?The classical O3-AHT, usually
consisting of 225 ml of blood (plus 25 ml of 3.8% sodium
citratesolution) treated with 225 ml of gas (O2_/O3) with ozone
concentrations ranging from an initial20 mg/ml slowly scaled up to
40_/50 mg/ml per ml of blood, continued for several months,twice
weekly, is ideal for this purpose. A probable explanation is that,
after each bloodreinfusion, a small percentage of immune cells are
activated and home in several organs: thesecells release into the
microenvironment cytokines that, in turn, prime or activate
neighboringcells thus slowly reinforcing immune responses.
Modifications of cytokine plasma levels arehardly detectable so
that side effects like the flu-like syndrome, typically observed
afteradministration of immunoadjuvants, are absent [39] and
actually most of patients report asense of well-being during the
therapy. There is a wealth of experimental data [40 - 44]
showingthat both animals and plants can develop ozone tolerance by
upregulating the expression ofantioxidants, which can correct a
chronic imbalance between excessive endogenous oxidationdue to
viral infections, cancer, chronic inflammations and depressed
antioxidants. Both chronichepatitis C virus and cancer patients
have shown a marked improvement of their clinicalconditions after
several months of O3-AHT treatments, suggesting that this
calculated andbrief oxidative stress truly merits the term
therapeutic shock.
3.3. PlateletsIt is known that ROS can induce platelet
activation and it was obvious to assume that bloodozonation, by
generating H2O2, could cause it. [45 46] Moreover H2O2 or other ROS
canactivate phospholipase C, phospholipase A2, cyclo-oxygenases and
lipo-oxygenases andthromboxane synthetase, allowing a step increase
of intracellular Ca2_, release of prostaglandin E2, prostaglandin
F2a and thromboxane A2 with irreversible platelet aggregation.
Forthese reasons it is studied the behavior of either human
platelet rich-plasma anticoagulatedwith heparin or citrate, either
untreated or simply oxygenated, or ozonated at three concen
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trations (20, 40 and 80 mg/ml). Because the plasmatic Ca2_ level
potentiates the ozone effect,we were not surprised to observe a
rapid platelet aggregation in heparinized plasma particularly at
the highest concentration. [47] Consequently the release of several
growth factors likeplatelet-derived growth factor AB, TGF-b1,
interleukin-8 and thromboxane_/2 were significantly higher from
heparinized platelets than Ca2_-free platelets. [48] These results
taught usthat it is better to chelate Ca2_ for performing a safe
autohemotherapy. Nonetheless the releaseof growth factors from
Ca2_-free platelets is still important because the reinfusion of
ozonatedblood implies an elevation of plasma levels of TGF-b that
may explain why the healing ofnecrotic ulcers in hind limb ischemia
due to atherosclerosis and diabetes markedly quickensduring
treatment with both parenteral (O3-AHT) and topical treatments with
ozonated waterand oil. [49 50]
3.4. Endothelial cells and the vascular systemDuring the
reinfusion of ozonated blood, the endothelium comes in contact with
traces of LOPsthat soon disappear in vivo. It is [51] investigated
the effect of addition of ozonated (40 and 80mg/ml) human plasma to
human endothelial cells in culture and it is measured a
significantincrease of the critical relaxing factor NO+that was
ozone dose dependent.The induction of nitric oxide synthase and the
release of NO+was reinforced in the presenceof 20 mm of arginine
and was abolished by the addition of 20 mM of
L-N-omega-nitro-L-arginine methyl ester. In physiological
conditions the endothelium regulates the vascular tone[52] by
producing some 1_/10 mM of NO+and 1 nM of anion superoxide (one of
the contractingfactors). The intravascular half-life of NO+is about
2 msec with a strictly localized consumptionso that the likelihood
of improving vasodilation in remote ischemic areas (the macula or
thelimbs) seems negligible. However, NO+readily reacts with GSH,
cysteine, albumin andhemoglobin (cysteine residue b 93) and the
formed S-nitrosothiols and S-nitrosohemoglobinhave half-lives of
5_/50 min, allowing a pharmacological effect at distant sites. [53
54] Itremains to be ascertained whether ozonated blood enhances the
release of prostacyclin (PGI2)and angiopoietins, both important
factors for improving ischemic vasculopathies.
3.5. Parenchymal cells in other organsUpon reinfusion of
ozonated blood, LOPs can reach other organs such as the
hypothalamus,endocrine glands, liver, kidneys and bone marrow. The
phenomenon of adaptation to therepeated and acute oxidative stress
imposed by O3-AHT is most interesting and able to elicitcrucial
therapeutic responses. During prolonged treatment, cells throughout
the body receivesmall and gradual pulses of LOPs that are
responsible for:1. Neuro-endocrine responses explaining the
reported feeling of wellness, [55]2. The upregulation of
antioxidant enzymes in several cell types that is an excellent way
to
re-equilibrate the oxidant_/antioxidant unbalance3. Inducing a
number of stress or heat shock proteins (HSPs) such as HSP27, HO-1
(HSP 32),
HSP72 and HSP [90 56 59]
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It is observed that HO-1 is a protective enzyme allowing the
formation of Fe2_, bilirubin (anantioxidant) and carbon monoxide
(CO), a vasodilator that, like NO+, increases the level ofcyclic
guanosine monophosphate, the reaction catalyzed by guanylate
cyclase. Besides gasesproduced by the gut flora, it is truly
remarkable that cells can release other gaseous molecules(NO+, CO
and CO2), and it is even more surprising that even ozone can be
produced byactivated antibody-coated neutrophils. [60] These gases
can now be considered as moleculesable to deliver crucial
physiological and pharmacological effects.Excessive amounts of
these molecules are toxic, causing serious pathological events
andpossibly death. Nature teaches us that these gases, depending on
their concentrations, can beeither friends or foes and similarly
ozone therapy can be either useful or toxic. If this reasoningis
correct, ozone therapy, when judiciously performed, is a simple,
inexpensive and atoxicapproach with the advantage of activating
several biomechanisms in different cells unusuallyleading to an
integrated and often incredible response.
4. The extreme versatility of ozone therapyThe sarcastic comment
of the opponents is that ozone therapy looks like a panacea for
alldiseases. Indeed it seems so, but in reality this is due to the
multitude of compounds originatedat first from the reaction of
ozone with body fluids, and eventually able to display
pleiotropiceffects delivered by different organs. For the sake of
brevity we can only summarize thetherapeutic effects so far
reported
Specialization PathologyDermatology Herpes Zoster and simplex,
acne, eczema, lipodystrophy (cellulite), mycosis, psoriasis,
atopic
dermatitisInternal Medicine Hepatitis, diabetes,
atherosclerosis, arterial hypertension, osteoarthritis, asthma,
chronic
bronchitis, gastritis, gastric ulcer, Crohn's disease, chronic
constipation, hypothyroidism.Nephrology / Dialysis Adjuvant in the
treatment of ischemic-metabolic pathologies.Neurology Migraines,
depression, vasomotor cephalea, neuro-vascular disorders.Dentistry
Treatment of cavities, disinfection of cavities during surgery and
post-operatory period.
Periodontitis, aphthas.Orthopedic Rheumatology Disc-radicular
conflicts, disc herniation, articular rheumatism, lumbago,
osteoarthritis,
arthropathy, periarthritis, rheumatoid arthritis.Angiology
Venous insufficiency, diabetic ulcer, arthropathy, coronaropathy,
gangrene, postphlebitic
ulcer, peripheral vasculopathy.Gynecology Bacterial infections
by protozoa or mycosis, Bartholin's cyst, vaginitis, menopause,
chronic
pelvic inflammation, infertility.Immunology Immuno-modulator,
autoimmune disorders, adjuvant in treatments with radiation and
in
immunodeficiency.
Table 1. Indiciations of Ozone Therapy in General
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4.1. Acute and chronic bacterial, viral and fungine
infectionsIntuitively, ozone therapy is very useful in both acute
and chronic bacterial, viral and fungineinfections because the
generated ROS are the natural and most effective agents to which
evenantibiotic resistant pathogens do not resist. [3-61] Moreover,
improvement of metabolism andimmunological functions contribute to
a favorable outcome. Abscesses, anal fissures, fistulae,bed sores,
furunculosis, inveterate osteomyelitis, vulvovaginitis, necrotizing
fasciitis andtorpid ulcers of various origin have been shown to
improve rapidly, particularly using thecombination of O3-AHT with
topical treatment using either direct O2_/O3 exposure or
thecleansing and stimulating effect of ozonated water and oil. The
activity of ozonated solutionsin eliminating the infectivity and
enhancing healing is almost unbelievable. However, inWestern
countries accustomed to the use of antibiotic creams (often with
corticosteroids) thereis no mental attitude to profitably use the
inexpensive and most active ozonated oil. [62]
4.2. Ischemic diseasesChronic limb ischemia (atherosclerosis,
diabetes, Burgers disease) is most effectively treatedat stage II-b
with complete disappearance of pain and claudication. Moreover,
since 1981,Rokitansky et al.[49] demonstrated that a cycle of
O3-AHT (usually 14 treatments) led to a verygood improvement in
70.6% and 53.8% of either stage III or stage IV (Fontaine)
patients,respectively. Amputation of toes and limbs could be
avoided in pre-terminal phases.These results have been amply
confirmed by Giunta et al.,[63] Mattassi et al. [64] and Tylickiet
al.[65] Preterminal patients with chronic heart ischemia and no
further susceptibility toconventional treatments have shown marked
improvement after a cycle of 14 treatments ofextracorporeal
circulation of blood against O2_/O3.[66] A randomized controlled
study is inprogress for establishing the validity of this more
invasive method than classical O3-AHT.
4.3. Age-related macular degenerationA 6-year study in 90
patients with the dry form of ARMD has been carried out performing
acycle of 13_/14 O3-AHT treatments. Mean distance best-corrected
visual acuity was significantly improved in the treatment group of
patients while in the control group, first treatedwith oxygenated
autohaemotherapy, only a modest and not significant improvement in
meandistance visual acuity was observed. No adverse effects have
been noted and the patientscompliance has been excellent. [26]
Owing to the constant increase of ARMD patients and thelack of an
effective conventional treatment, this approach appears
mandatory.
4.4. Orthopedic diseasesUntil recently it was unthinkable that a
mixture of O2_/O3 could be useful in orthopedics.Indeed lumbar disk
herniation and osteoarthritis, although having different
etiologies, have acommon inflammatory background expressed by a
localized chronic oxidative stress due toexcessive production of
ROS, release of proinflammatory cytokines and activation of
cycloxygenases. Common sense would proscribe the use of ozone, a
master generator of free radicalsand, as it is well shown, [34 67]
after pulmonary exposure, a superb inflammatory agent.
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Contrary to all expectations, it is now well demonstrated [68]
that combined intradiscal andperiganglionic injection of medical
ozone allows an excellent outcome in 70.3% of patientstreated for
disk herniation performed after conservative management failed to
respond. In thesame vein, it appears very surprising that the
application of medical ozone in acute and chronicpainful diseases
of the joints allows rapid pain relief, disappearance of
inflammation andimprovement of mobility. Thousands of patients have
been successfully treated and the lackof side effects is
noteworthy. [69] These positive empiric observations need to be
explained.Ozone is indeed a surprising gas that paradoxically,
after prolonged administration at lowconcentrations, induces
tolerance, a phenomenon termed hormesis by Goldman [70] toindicate
a beneficial effect of a low level exposure to an agent that is
harmful at high levels.Thus, at this stage, we use the definition
of ozone paradox for explaining these excellenttherapeutic results.
Immediately after O2_/O3 administration in the nucleus pulposus or
intoinflamed endoarticular cavities; a sort of oxidative shock
seems to subvert all the traditionalrules by inducing an
antioxidative response due to several factors, among which is
thecholinergic antiinflammatory pathway. [71] A detailed discussion
is reported elsewhere. (3)
4.5. DentistryThis is another medical specialty where ozone has
been recently evaluated with exceedinglyinteresting results.[72]
Primary root carious lesions are being treated with a novel
ozonedelivery system able to avoid any toxic risk for the mouth
cavity and lungs. The tooths lesionis exposed for 10_/20 sec to a
sort of ozone hurricane based on a gas flow of 615 ml/min ofO2_/O3
at a low concentration (4 mg/ml), perfectly enclosed in a tightly
fitting silicone cupenclosing the tooth. It is not surprising that
all bacteria, particularly lactobacilli, are destroyedso that the
ozone-sterilized dental surface becomes quickly remineralized,
becoming hard andresistant to further bacterial attack. This new
approach is simple, inexpensive and welltolerated, as opposed to
the conventional and painful drilling and filling management
ofprimary root carious lesions. Dermatological, pulmonary, renal,
hematological and neurodegenerative diseases Owing to the ability
of ozone to activate a number of biological targets,ozone therapy
could be proficiently used in some dermatological, pulmonary,
renal, hematological and neurodegenerative diseases. However these
pathologies so far have not beenevaluated in a controlled fashion.
Most of the patients with metastatic cancer resistant
toradiotherapy and chemotherapy report a striking improvement of
the quality of life withprolonged (twice weekly for months) O3-AHT
treatments. [25 73] This is a constantlyobserved result, most
probably due to a multifactorial neuroendocrine response.
5. SummaryFinally it must be emphasized that if ozone is
judiciously used according to precisely definedguidelines, it
causes neither acute, nor chronic side effects. After two decades
of practicalapplications and the results observed in patients after
conventional remedies have provedunsatisfactory. One has the
feeling that, if ozone therapy could be accepted and used in
all
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hospitals, it would represent a small but important medical
revolution able to cure or stabilizeseveral diseases in many
patients in both rich and poor countries.
6. Discussion and conclusionsThere is no doubt that ozone can be
toxic, and even today its hazardous employment bycharlatans and
unprepared physicians has contributed to a poor consideration of
ozonetherapy. That is one reason why the use of ozone is prohibited
in some states of the USA andwhy this therapy is still regarded
with skepticism by orthodox medicine even in Germany,where this
approach was first conceived. Moreover, the following data tend to
generalize thatozone is always toxic and should not be used in
medicine:1. Overwhelming evidence shows that the
bronchial-pulmonary system is very sensitive to
ozone and this gas should never be inhaled. [67]2. This is very
true because the respiratory tract lining fluid is constituted by a
very thin,
watery film containing a minimal amount of antioxidants that
makes mucosal cellsextremely vulnerable to oxidation. The opposite
situation occurs for blood cells suspendedin plasma, which has a
potent antioxidant capacity (1.23_/1.83 mmol/l) able to tame
anyozone dose within the therapeutic range (10_/80 mg/ml).
3. Saline-washed erythrocytes suspended in saline undergo
extensive hemolysis after ozoneexposure. [12]
4. Cells in culture, even if exposed to very low ozone
concentrations for a long time, undergoapoptosis. [74]
5. One-hour exposure of saline-diluted blood to 5 mM of ozone
induces genotoxic effects onleukocyte. [75]
But is ozone always toxic?As a matter of fact millions of
O3-AHT, even if performed in an empirical fashion during thepast
three decades, has neither yielded acute nor chronic toxic effects.
According to Jacobs [76]this procedure has yielded the lowest
number of medical complications.However, four deaths have been
recorded due to pulmonary embolism, which occurredduring direct
intravenous administration of O2_/O3, an application prohibited by
the European Society of Ozonetherapy since 1983. Thus ozone seems
like Janus and his two facesrequire an explanation. This is now
reasonably clear. Since 1988 we have investigated theproblem in a
scientific way using precise ozone generators, which allow checking
ozoneconcentration in real time by a photometer calibrated with the
classical iodometric method. Areview [61] and a critical book (3)
have extensively clarified the issue but this does not
seemsufficient to dispel the dogma that ozone is always toxic.
However, we now consider ozoneas a real drug that must be used with
caution after having carefully defined its therapeuticwindow.
First, the ozone must be calibrated against the antioxidant
capacity of the patientsblood in order to control the potential
ozone toxicity
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Second, expert scientists in free radicals ought to distinguish
the chronic intracellular oxidativestress typical of several
pathologies by the transitory (5 min) calculated oxidative
stressoccurring when a precise volume of blood is exposed ex vivo
to an equal volume of gas (O2_/O3) with well-defined ozone
concentrations ranging from 20 up to 40 mg/ml per ml of blood.It
needs to be emphasized that the exogenous oxidative stress caused
by ozone in blood is dueto the fact that ozone, once dissolved in
the plasmatic water, instantaneously reacts withbiomolecules and
disappears but generates ROS, among which are H2O2 and LOPs. These
arethe effective ozone messengers that interact with a variety of
cells and elicit the now-termedtherapeutic shock due to the
multiform biological responses. That ozone acts as a realchemical
drug is proved by the fact that the ozone messengers, to be
effective, must reach athreshold because otherwise there are no
biological effects and the therapeutic results, if any,are due to a
placebo effect. Although we have proven that ozone therapy is not a
nebulousapproach and has been shown amenable to a precise
scientific scrutiny, it is probable that muchstill remains to be
uncovered.Everyone knows that plasma and blood cells contain an
almost redundant antioxidant systemmade up of hydro-liposoluble
compounds and antioxidant enzymes. During aging or pathologic
conditions, this is not sufficient to correct the intracellular
oxidative stress, but normallyit is adequate to tame ozone toxicity
while allowing the generation of ROS and LOPs. Thus alldata
emphasizing ozone toxicity can be easily dismissed because the
following is now wellproven:1. Blood is a much more ozone-resistant
tissue than the respiratory tract that, for anatomic,
biochemical and metabolic reasons, is always at a loss when
exposed to ozone, andtherefore it is wrong to extrapolate ozone
toxicity for the pulmonary system to blood.
2. Washed and saline-resuspended erythrocytes, fully depleted of
the plasmatic antioxidants, are obviously very sensitive to
ozonation, and all of these unnatural data haveneither
physiological nor practical significance.
3. The same occurs for cells cultured in antioxidant-poor media
and exposed continuouslyfor days to ozone. Surprisingly, cell
biologists reported only the ozone concentration buthave neither
calculated nor taken into account the cumulative dose of ozone that
after along exposure kills the cells.
4. The conclusion is that, although ozone is potentially
mutagenic, so far all experimentaldata performed in physiological
conditions and clinical evidence have neithershown anycell damage
nor adverse effects in patients. As a matter of fact, blood is
exposed to ozoneconcentrations (0.21_/1.68 mM) lower than the
mutagenic ones (1.5_/5.6 mM). Thequestion of whether ozone is
genotoxic and mutagenic is a critical one and has beenextensively
discussed elsewhere. (3) What has never been entirely appreciated
is the factthat we can only use an ozone dose that does not
overwhelm the antioxidant capacity ofblood.
Hopefully this discussion should put an end to the confusion
between the endogenouslyconstant oxidative stress due to the oxygen
and the transitory and occasional therapeuticshock due to precise
blood ozonation. A point that should not be overlooked is that
ozone
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messengers, by acting on different cells, elicit a variety of
biological effects that cannot ever bedreamed of with the usual
reductionist approach of using a drug for a single target.
Thisconsideration can explain the far superior therapeutic effect
of parenteral and topical ozonetherapy in advanced cases of chronic
limb ischemia to the conventional infusion of prostanoids.Another
relevant characteristic is that the judicious strategy start low,
go slow in using ozoneis able to induce in patients the adaptation
to the chronic oxidative stress (i.e. ozone) paradoxically
upregulates the antioxidant defenses. The scientific evaluation of
ozone therapyefficacy remains the crucial point: results accrued
during the past 20 years show that is veryuseful in chronic limb
ischemia, ARMD, chronic infectious diseases and, most surprising,
inorthopedics and even in dentistry after conventional medicine has
failed to provide a realadvantage. There are no adverse effects and
most of the patients report a feeling of wellness.The efficacy
remains uncertain in other pathologies such as neurodegenerative,
autoimmunediseases and cancer because clinical experience is
fragmentary and anecdotal.However, orthodox medicine remains
skeptical because controlled clinical trials are few andare not
considered satisfactory. Unfortunately our good will is not
sufficient to overcomeprejudice and lack of sponsors. It is
distressing to realize that a wrong dogma, commercialinterests and
the disinterest of Health Authorities delay the application of a
medical approachthat could help billions of patients, particularly
in poor countries. Finally, this paper may servethe purpose of
opening a fruitful discussion on the beneficial versus the
toxicological actionsof ozone, and a referee has proposed that the
debate may be hosted as a forum by Mediatorsof Inflammations.
Author detailsRuhi CakirMediozon Clinics, Istanbul, Turkey
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